Tetracycline recovery



May 12, 1959 B. HEINEMANN EI'AL TETRACYCLINE RECOVERY 2 Sheets-Sheet. 1

Filed Sept. 50. 1958 GOP PERCENT TRANSMISSION oow com 82 oo= com. 009

JOHN P. MURPHY ATTORNEY May 12, 1959 B. HEINEMANN ETAL TETRACYCLINE RECOVERY 2 SheetS-Sheet 2 Filed Sept. 50, 1958 00% Dow OON Oom-

O 7 O Q 'd PERCENT TRANSMISSION INVENTORS BERNARD HEINEMAN N IRVING R. HOOPER BY JOHN P. MURPHY ATTORNEY United States Patent TETRALCY'CLINE RECOVERY Bernard Heinemann, De Witt, and Irving R. Hooper, Fayetteville, N.Y., assignors to Bristol Laboratories Inc., East Syracuse, N.Y., a corporation of New York Application September 30, 1958, Serial No. 764,284

2 Claims. (Cl. 260-559) This invention is concerned with novel and useful methods for the production of an antibiotic we have called Omegamycin, and which is-now more commonly called tetracycline. More particularly, it relates to particular methods for its recovery and concentration from crude solutions including the fermentation broths and purification thereof.

This appliaction is a continuation-in-part of our prior copending application Serial Number 432,388, filed May 26, 1954, now abandoned, which was in turn a continuation-in-part of prior copending applications Serial Numbers 388,048, filed October 19, 1953 and now abandoned, 404,380, filed January 15, 1954, and now abandoned, and 406,062, filed January 25, 1954 and now abandoned.

During the past few years a number of metabolic products of the growth of bacteria and fungi have been isolatedand found to possess valuable therapeutic properties. Among these may be mentioned penicillin, streptomycin, gramicidin, tyrocidin, bacitracin, 'subtilin, streptothricin, Aureomycin (chlortetracycline), Terramycin (oxytetracycline) and others. Some of these have proven to be extremely valuable because of their elfectiveness against pathogenic organisms. Others have been found to be of limited usefulness for various reasons, as because of their toxicity. Chlortetracycline, oxytetracycline and tetracycline have been particularly useful because of their broad spectrum of activity. Of these, tetracycline is an antibiotic of broad spectrum which gives better blood levels and fewer side reactions than chlortetracycline and oxytetracycline and in particular is more stable than chlortetracycline in alkaline media.

The object of the present invention is to provide improved methods of preparing tetracycline which are suitable for commercial use.

In accordance with the present invention there is provided a process for recovering tetracycline from a fermentation broth comprising the consecutive steps of adjusting the pH of an acidic fermentation broth to a pH in the range of 8.5 to 9.4 inclusive, extracting the tetracycline with n-butanol, separating the n-butanol and recovering the tetracycline therefrom.

The tetracycline produced by the process of the present invention is an amphoteric substance effective in inhibiting the growth of Gram-positive and Gram-negative bacteria and. capable of forming salts with acids and Patented May 12, 1959 metals, whose anhydrous base-has the empirical formula C H N O ,m elts substantially at l-173 C. with decomposition, has [u] 3 245 at a concentration of 1% in methanol, has ultra-violet absorption peaks at 267 m (as, 17,400) and 355 my. (6, 13, 500) in 0.1 N hydrochloric acid and exhibits when. suspended in mineral oil characteristic absorption in the infrared region of the spectrum at the following frequencies expressed in reciprocal centimeters: 3490, 1672, 1607, 1524, 1259, 1222, 1130, 1065, 1040, 990, 963, 948, 932, 861, 838, 803, 786, 741, 739, and 668; its hydrochloride melts substantially at 217-219 C. with decomposition, has the empirical formula C22H24N208'HC1, has [a] 253 at a concentration of 0.5% in 0.1 N hydrochloric acidand exhibits when suspended in mineral oil characteristic absorption in the infrared region of the spectrum at the following frequencies expressed in reciprocal centimeters: 3340, 1678, 1623, 1597, 1315, 1248, 1229,1175, 1140, 1061, 1036, 1002, 964, 949, 864, 823, 796, 781, 743, 719, 692, and 667; and its formate melts substantially at 176-177 C. and has the empirical formula Tetracycline exerts 'an inhibiting action on the growth of many bacteria, as described in detailbelow.

In accordance with the present invention, tetracycline is prepared among other methods by the cultivation under particular controlled conditions of a hitherto undescribed species of micro-organism which We have tentatively called Streptomyces BL 567201 which was isolated from a sample of soil. A culture of the living organism which has been isolated from this soil and identified as Streptomyces virz'difaciens has been deposited in the American Type Culture Collection, Washington, D.C. and added to its permanent collection of microorganisms as A.T.C.C. 11989. The description of this organism is set forth in the following.

The organism Slreptomyces viridifaciens which produces tetracycline belongs to the genus distinguished as Streptomyces. Growth of this organism is good on glycerol-asparagine-beef extract agar at 30 C. On this medium mouse-gray aerial hyphae are formed-and a yellowish-green pigment is secreted into the agar medium. The mycelium is composed of branched hyphae, the younger elements of which are Gram-positive. Condida are produced on aerial hyphae.

The above color descriptions are from: Dictionary of Color; by Maerz and Paul, 1st edition.

It is to be understood that for the production of tetracycline we do not wish to limit ourselves to this particular organism or to organisms fully answering the description herein which is given merely for illustrative purposes. We especially wish to include the use of organisms which are mutants produced from the described organism by mutating agents such as X-radiation, ultraviolet radiations, nitrogen mustards, etc.

Tetracycline is a valuable therapeutic agent, e.g. in human or veterinary medicine. Tetracycline possesses the particular advantages of a broad spectrum, high blood levels and low toxicity. Tetracycline exhibits highly use ful resistance to degradation by heat or water in either acid or alkaline media. Certain metal and acid-addition salts of tetracycline have been found to be even more useful than tetracycline base by virtue of their decreased hygroscopicity and increased solubility in water.

The antibiotic, tetracycline, has been found to be active in vitro against a number of Gram-positive and Gram-negative bacteria. The following chart shows the antibiotic activity of Omegamycin (lot 25), Aureomycin HCl, Terramycin HCl, and tetracycline when placed in a trench cut in a plate of heart infusion agar (pH 7.0):

PLATE SPECTRUM [5 mg./mi.]

Zone of Inhibition in mm.

Tetracy- Tetracycline. cline.

Organism (Isolated (Prepared Aureo- Terra- Fermentaby Dechlornycin myoin tion broth.) rinating (3 mg./ (1 mg./

Lot 25. Chlortet- 1111.) m1.) (5 mgJml.) racycline.) (5 mg./ml.)

6 8 11 9 7 8 8 10 5 8 10 11 9 11 11 13 S. paraiyphi 10 12 13 15 S. pullorum- 8 11 11 13 A. aerogenee... 5 6 9 11 Pa. fluorescens 1 7 9 13 A20. fecalis. 6 11 8 8 Pr vulgarz's- 6 4 O V cholerae 8 11 11 14 Neisseria sp. 8 7 8 11 arerosis 11 14 14 27 M mycozdes. 10 10 16 27 B. cereus 10 11 13 17 S. marcescens. 0 2 2 0 tetragenus 11 15 27 27 S fleznerz'w 14 11 10 14 S dysenter 9 11 1O 15 C albz'cans 6 0 0 0 Staph. aureus. 10 13 13 18 E. typhosa 7 9 10 13 E. coli 4 7 11 11 S. paratyphi B" 9 11 16 14 K. pneumoniae 6 15 14 P8. aeruginosa... 1 4 9 7 S. gallinarum. 8 11 12 13 B anthracis..- 7 19 27 S schoitmullerz 10 12 12 B subtz'lz's 10 9 11 13 B mycoides O. 13 8 15 20 The spectrum test is performed as follows: Approximately ml. of sterile heart infusion broth (Difco), with 2% of agar added as a solidifying agent, is placed in a sterile Petri dish (3% diameter) and allowed to harden. A trench 8 mm. x mm. is then made in the agar with a sterile spatula. The bottom of the trench is sealed with a drop or two of melted agar. A streak is then made from a 24-hour nutrient broth culture of each test bacterium, previously incubated at 37 C., with a small loop, streaking from the edge of the trench to the wall of the Petri dish. The trench is next filled with a 5 mg./ ml. solution of the antibiotic. The dish is then placed at 37 C. for 18-24 hours. A linear measurement of the zone of inhibition is made from the edge of the trench to the point where growth of the test organism occurs, after the selected number of hours within the period indicated.

The following is a summary of antibacterial activity tests in vitro with crystalline tetracycline hydrochloride and three other antibiotics. The test used was a tube dilution technique and the minimal concentrations of antibiotic completely inhibiting growth of the bacteria were determined. Heart infusion broth was used as the medium for all test organisms, except as noted.

Inhibitory Concentration, meg/ml.

Organism Tetracy- Ohlor- Oxy- Chloramcline H01 tetracytetracyphenicol cline HCl ciine H01 Micrococcus pyogenes var.

aureus 0. 15 0. 25 0. 25 5.0 Gaflkz a tetragena 0. 03 0. 06 0. 13 5. 0 Streptococcus pyogenes C203 2. 5 0.06 0.06 0.63 Streptococcus agalactiae 7077.- 0. 3 0.13 0. 13 1. 25 Streptococcus dysgalactiae 0926. 1. 25 0. 25 0. 13 1. 25 Streptococcus uberis 0. 13 0. 13 2. 5 Diplococcus pneumoniae 0. 15 0. 06 0.06 1. 25

Lactobac'lllus acz'dophilus #4356 2 0.6 0. 50 1. 0 2. 5 Lactobacillus 041862 #4646 2 1. 25 0. 50 1. 0 5.0 Lactobacz'llus lelchmann 2. 5 0. 50 1. 0 2. 5 Bacillus anthraczs 0. 0375 0. 02 0. 03 2. 5 Bacillus cereus v 0. 0375 0. 02 0.03 2. 5 Bacillus subtilis 5.0 1.0 1. 0 2. 5 Coryncbacterz'um rerosis 0.6 0.25 0.25 0. 63 Clostridium welchii 601 0.06 0.06 0.13 6. 25 Olostridium welchii M 3 0.25 0. 13 0.25 6. 25 Clostridium sporogenes 84 0. 125 0.25 0.25 6. 25 Clostridium sporogenes 40 0.06 0. 06 0.13 6. 25 Salmonella typhosa 2. 5 0.39 0. 78 1. 56 Escherichia coli 2. 5 1. 56 1. 56 12. 5 Shigella sonnei 1. 25 0.39 0.78 1. 56 Klebsiella pneumoniae- 2. 5 0. 78 0. 78 1. 56 Proteus vulgaris 10.0 12. 5 25. 0 6. 25 Pseudomonas aeruginosa 10.0 6. 25 1. 56 50.0 N eisseria sp 5 0. 19 1. 56 50. 0 Candida albicans #520.- 0 100 50 1 Assayed in 10% serum broth. 1 Assayed in tomato juice broth. Assayed in thioglycoilate broth.

The following is the diffusion plate assay method for determining the activity of tetracycline.

Culture Medium Streptomycin Assay Agar (with yeast extract) was purchased from the Baltimore Biological Laboratories, Baltimore, Maryland, and used as directed on the label. A suitable preparation may be made by suspending in one liter of distilled water to a final pH of 6.2 or 8.0 a mixture of 1.5 g. beef extract, 3 grams yeast extract, 6.0 grams peptone (e.g. Gelysate) and 15 g. agar. The suspension is allowed to stand for five minutes, mixed until a uniform suspension is obtained and heated gently with stirring. The suspension is boiled one or two minutes or until solution has occurred. The culture medium is then dispensed and sterilized at 121 C. (fifteen pounds per square inch of steam pressure, gauge, for fifteen minutes).

Inoculum The test organism is Bacillus subtilis American Type Culture Collection 6633. A spore suspension containing 5 0,000,000 viable spores per ml. is added to the abovedescribed melted assay agar (cooled to 53 C.) to give a final inoculum of 2%.

Preparation of plates Buifer at pH 6.2 or 8.0 as desired is used for making dilutions. Thus at pH 6.2 a citrate buffer is used for making dilutions. This is prepared by mixing 192.12 g. of anhydrous citric acid with 106.3 g. sodium hydroxide in a liter of distilled water and diluting the mixture to one-tenth concentration with distilled water. The pH of the buffer must be checked potentiometrically, and,

ifnecessary, adjusted to pH 6.2 by'the addition of citric acid or sodium hydroxide. Variations in pH or concentration of the buffer affect the sizes of inhibition zones markedly. It has not been found necessary to sterilize the buffer. The stock solution is preserved with chloroform or toluene and fresh working solutions prepared daily.

At pH 8.0 a phosphate buffer is used for making dilutions. This is prepared by mixing 95 ml. of molar K HPO with 5 ml. of molar KH PO and diluting the mixture to one-tenth concentration with distilled water. The pH of the buffer must be checked potentiometrically, and, if necessary, adjusted to pH 8.0 by addition of one or the other phosphate solution. Variations in pH or concentration of the buffer affect the sizes of inhibition zones markedly. It has not been found necessary to sterilize the buffer. The stock solution is preserved with chloroform or toluene and fresh working solutions prepared daily.

Assay Unknown samples are diluted, if required, in the buffer. Three holes on each plate are used to receive a single dilution of the sample. Following incubation at 32 C. the diameters of the zones are measured and averaged.

Sta'eptomyces viridifacz'ens was differentiated from a strain of S. aureofaciens (NRRL 2209) obtained from the Northern Regional Research Laboratory, Peoria, Illinois, Where it had been deposited as an authentic aureomycin- Streptomyces Streptomyces Medium aureofaciens viridifaciem N RRL 2209 Nutrient agar Good growth. Pro- Good growth. No

Asparagine-meat extract-dextrose agar.

Potato slants Litmus Milk duction of aerial hyphae and spores is inhibited somewhat and is white to gray in color. Straw soluble pigment.

Good growth. Abundant aerial myeelium and spores, cement gray to frost gray in color. India bnfi soluble pigment.

Growth raised, surface nodulate, India butt color.

Neither significant pH change nor apparent peptonization in 15 days.

aerial mycelium, colony tan to light brown. Cinnamon soluble pigment.

Good growth. Abundant aerial rnycelium and spores, gull colored, India bufi soluble pigment.

Growth raised, surface nodulate, earn-beige color.

Alkaline with peptonization. Very good growth.

This invention embraces a process for growing species producing strain, by observation of growth characteristic on glycerol asparagine beef extract agar and Czapek- The agar mixtures DoX agar containing 1% dextrin.

employed and the results obtained were as follows:

GLYCEROL ASPARAGINE BEEF EXTRACT AGAR Percent Glycerol 1 Asparagine 0.05 Beef extract 0.2 K HPO 0.05 Agar 1.5 Sterile water, q.s. 100%.

Streptomyces Streptomyces viridifaciens. aureofacz'ens Growth. Goo Good. Spornlation do Do. Difiusible pigment. Yellowish-green-.- None. Spiral formation... Abundant, loosely Do.

wound. Aerial Hyphae-- Mouse-gray Rose-gray. Reverse Brown Olive-drab.

DEXTRIN CZAPEK-DOX Percent NaNO 0.2 K HPO 0.1 MgSO.; 0.05 KCl 0.05 FeSO Trace Agar 1.5 Sterile water, q.s. 100%.

Streptomycer Streptomyces viridifaciens uureofaciens Growth Fair to good Fair. Sporulation ood Poor. Difiusible pigment None None. 7 Spiral formulation Abundant, loosely Sparse, very loosely wound. Wound. Aerial hypliac Mouse-gray Butt to gray. Reverse Light brown Buff to tan.

The Streptomyces viridifaciens is further characterized of microorganisms at about 24-30 C. under submerged conditions of agitation and aeration on media consisting of sterile water containing a source of carbon, a source of nitrogen, at source of growth substances, mineral salts such as sodium chloride, potassium phosphate, magnesium sulfate, sodium nitrate and, when desired, a buttering agent such as calcium carbonate.

As a source of carbon in the nutrient medium carbohydrates are satisfactory. Any of the following can be used.

Ordinary starch Xylose Soluble starch Arabinose Sucrose Rhamnose Glucose Fructose Maltose Lactose Dextrose Inulin Glycerol Dextrins Galactose Mannitol These carbon sources are supplied to the medium in purified form or in the form of concentrates. The amount of such carbon sources for best antibiotic production in the medium varies considerably, from about /2% to 5%, by weight of the total weight of the fermentation medium.

Suitable sources of nitrogen, including some sources of growth substances, and including organic and inorganic nitrogen-containing compounds and proteinaceous .materials in particular, for the fermentation process include a wide variety of substances such as:

Amino acids Casein, both hydrolyzed and unhydrolyzed Fish meal Soy bean meal Meat extracts Liver cake Urea Nitrates Ammonium compounds Distillers grain slop Corn-steeping liquor Wheat-steeping liquor Whey or whey concentrates Acid hydrolyzed corn gluten Acid hydrolyzed wheat gluten Peptone Olfals Brewers yeast Cottonseed meal Lactalbumin Tryptone Palm oil meal Coconut oil meal Linseed oil meal Peanut oil meal Sunflower oil meal These proteinaceous ingredients need not be applied in a high degree of purity; the less pure materials which carry traces of growth factors and considerable quantities of mineral nutrients are suitable for use. It is not possible, of course, because of the crude nature of many of these nitrogenous substances to specify definite proportions of the material to be added. An amount of about 0.1% to 5.0% by weight on a solid basis describes the useful range of nitrogenous substances to be added to the media in most cases. Particularly high yields are obtained by the use of media containing soy proteoses or soy peptones, that is, degraded soy bean products, including hydrolyzed soy bean meal.

Among the mineral salts used in the medium are certain heavy metal salts useful in trace amounts and often found to such extent in crude, natural components of the media, such as corn steep liquor. When not present in other components of the media, these salts are added and found to increase the yield of tetracycline. Thus, sufiicient manganese, copper and particularly zinc are introduced to insure that the amounts present both as impurities in other material and by addition are at least approximately 0.00033% manganese as MnCl -4H O, 0.00033% copper as CuSO -5H O, and 0.003% zinc as ZnSO -7H O.

The pH of the fermentation medium should be about 5.0-6.5 and preferably 5.8-6.2 at the start of the fermentation. The preferred temperature of the fermentation process is about 2430 C. The maximum yield of product is usually obtained in 1-5 days, varying with the method of cultivating the Streptomyces.

Omegamycin (tetracycline) is active in vivo as well as in vitro and displays marked chemotherapeutic activity against experimental infection in mice. The results of such testing and of determinations of toxicity are given in the following table.

The CD (Curative Dose-50) is the minimum dose of Omegamycin which will cure 50 percent of a group of mice injected intraperitoneally with 100 to 1000 LD doses of Diplococcus pneumoniae, each LD dose being sutficient if given alone to kill 50 percent of a group of mice. The infection is given at once after the second dose of the test drug. The test drug is given in two equally divided doses at approximately an 18-hour interval. The animals are observed for four days and deaths for each group expressed as the percentage of the total animals per group. The percentage death is transformed to probit values and these plotted against the log of the dose in mgm. per kg. of mouse weight. The point of intersection of the probit 5 line and the best line constructed through the experimental points indicates the concentration of drug which should protect half of the animals under the conditions of the experiment. The antilog of this term is called CD value.

EXAMPLE 1 In preparing tetracycline on a laboratory scale, the fer- Percent Sucrose 1 Soy bean meal 1 Soy peptone 1 KH PO 1.5 (NH HPO 0.5

Water, q.s. 100%.

are introduced into 4 liter bottles and sterilized. After autoclaving, the medium is inoculated with about 1%, by volume, of a turbid aqueous spore suspension of the Streptomyces from an agar slant. The pH is 6.0-6.2 at the start of the fermentation. The contents of the bottle are then incubated at 26-28 C. for 48 hours while shaking at 130 strokes per minute with a 1%" stroke. At the end of the incubation period the fermentation liquor was a bluish-green and when assayed by the method described above, gives inhibition zones of approximately 27 mm. when the broth is diluted 30-fold. This broth contains tetracycline which can be isolated as described below.

EXAMPLE 2 For larger scale production of tetracycline, an inoculum is prepared in a fermentation medium containing, by weight,

- Percent Corn steep 1 Sucrose 1 (NH HPO 0.5 KH PO 1.5 MgSO; 0.2 Water, q.s 100 made up in a volume of 2500 ml. and introduced into a 2 gallon bottle. The medium is sterilized with steam at l18120 C. for one hour. When cool, the medium is inoculated with about 0.5%, by volume, of a turbid, aqueous spore suspension of the Streptomyces from an agar slant. The contents of the bottle are then incubated at 26 28 C. for 48 hours on a reciprocating type shaker and sterile air blown over the surface of the liquid. From the inoculum bottle the broth containng the Streptomyces viridifaciens is transferred into the tank fermenter under completely aseptic conditions (see Example 3). When desired, the liquor may be processed as described below and the tetracycline isolated.

EXAMPLE 3 The antibiotic produced by Streptomyces viridifaciens, tetracycline, may be prepared on a large scale by submerged or deep culture. Stationary vat fermenters equipped with suitable agitation and aeration have been found to be useful for this purpose. A nutrient medium consisting of 56.8 liters of corn steep, 56.8 kg. sucrose, 28.4 kg. (NI-IQ HPO 85.2 kg. KH PO 11.3 kg. MgSO -7H O and water to make 1500 gallons is suitable. This medium may be prepared in a ZOOO-gallon glasslined fermenter equipped with a. water-jacket for tempera ture control, a suitable stainless steel agitator and a suitable sparging device for aeration. The medium is sterilized by heating with steam under pressure and is then cooled. After sterilization the hydrogen ion concentration of the medium should be approximately pH 6.2. The nutrient medium is inoculated with 15%, by volume, of a vegetative culture grown either in a similar type fermenter previously inoculated with an inocul'unr described earlier or with an inoculum prepared in the laboratory. The culture in the 2000 gallon fermenter is incubated at a temperature of 83 F. for two or three days. During the incubation the impeller is revolved at the rate of 90 rpm. and sterile air is introduced into the medium through the sparger at the rate of 100 cubic feet per minute. At the end of the incubation period the culture fluid normally contains sufiicient antibiotic activity to result in a zone of inhibition against the test organisms of approximately 23 mm. in diameter when a 16-fold broth dilution is tested by the assay procedure described above. The tetracycline is isolated as described below.

1 EXAMPLE 4 Fermentation liquor containing tetracycline is pro duced by following the procedure of Example 1, using the following culture medium:

Percent Cottonseed meal 1 Glucose 1 Distillers soluble 0.05 NaCl 0.5

CaCO 0.1 Water, q.s. 100

EXAMPLE 6 Fermentation liquor containing tetracycline is produced by following the procedure of Example 1, using the following culture medium:

, Percent Corn steep liquor 1 Cerelose 1 NaCl 0.5 CaCO 0.1 Water, q.s l 100 EXAMPLE7 Fermentation liquor containing tetracycline is produced by following the .procedure of Example 1, using the fol lowing culture medium: 1

Percent Soy bean meal 1 Cerelose 1 NaCl 0.5 Yeast extract 0.05 CaCO 0.1 Water, q.s 100 EXAMPLE 8 Fermentation liquor containing tetracycline is produced by following the procedure of Example 1, using the fol lowing culture medium:

- Percent Soy bean meal 3 Corn starch 0.5 N-Z-Amine B (Enzymatic digest of casein) 0.1 NaNO 0.3 CaCO 0.5 Water, q.s. 100

EXAMPLE 9 Fermentation liquor containing tetracycline is produced by following the procedure of Example 1, using the following culture medium:

After fermentation has been completed and as illustrated below by Examples 10 through 16, 18 through 22, 25, 27, and 29, tetracycline was formerly recovered from the broth, for example, by filtering to remove the mycelium, stirring the broth (preferably at about pH 8.5) with butanol or methyl isobutyl ketone, separating the solvent layer containing the tetracycline, concentrating it to small volume by distillation, and mixing it with a liquid lower hydrocarbon, e.g. Skellysolve C, to precipitate solid tetracycline as the base, if the extracted broth was at alkaline pH, e.g. pH 8.5, and as the hydrochloride, if, for example, the broth had been acidified before extraction with hydrochloric acid. The solid tetracycline so produced was further purified by slurrying into ammonium hydroxide and also by adsorption from a solution of the free base on to a chromatographic adsorbent (e.g., a silica such as Florisil), followed by washing (e.g., acetone or methanol) to remove impurities and then elution with an acid. The purified tetracycline acid salt in the eluate is then recovered by crystallization, precipitation, lyophilization, or the like. The liquor lower hydrocarbons used as precipitating agents are commercially available petroleum ethers, eg. Skellysolve A, B.P. 28-38 C.; Skellysolve B, B.P. 60-71 C.; Skellysolve C, 3.1. -100 C.

According to the improved processes provided by the present invention and after fermentation has been comleted, the tetracycline is recovered from the fermentation broth by an alkaline precipitation. Fundamentally, the fermentation broth is adjusted to an alkaline pH in the range of 8 to 11 inclusive and the precipitated mixture of mycelium (mat) and tetracycline is collected by filtration. As a variant of this process, the fermentation broth is acidified to less than pH3, filtered to remove the mycelium, and the filtrate is treated as above, that is, it is adjusted to an alkaline pH in the range of 8 to 11. inclusive and the precipitate of crude tetracycline is collected by filtration. Any water soluble alkali of suflicient strength is used, ammonia or sodium hydroxide are preferred. The pH is adjusted to the range of 8 to 11 inclusive; an alkaline pH in the range of 9 to 10 is preferred. The precipitate of of tetracycline, with or without accompanying mycelium is brought into solution for further manipulation as desired according to any of the procedures disclosed herein by solution in aqueous acid below pH 3 or in aqueous alkali above pH 11. Examples of such aqueous acids include water-soluble acids furnishing the desired pH, e.g. hydrochloric acid, sulfuric acid, phosphoric acid and nitric acid. These same acids are effective in lowering the pH of fermentation broth below pH 3, and preferably to about pH 2.0-2.5, to permit separation of tetracycline, which is in. solution as an acid addition salt, from the solid mycelium by filtration before use of the alkaline precipitation described above.

Various acid addition salts of tetracycline may be prepared, most simply by adding the desired acid, inorganic (including mineral acids) or organic, to the antibiotic in water until a clear solution is obtained. The solid salts may be prepared by adjusting the pH of such a solution of a tetracycline salt to a point just below that at which the antibiotic would begin to separate. The solution may then be dried, as by subjecting the frozen solution to a vacuum. Acids salts of tetracycline are obtained by evaporation of a solution of the salt in Water at a low pH. Mineral acids. which may be used are hydrochloric 11 acid, sulfuric acid and phosphoric acid. Organic acids which may be used are citric acid, tartaric acid, gluconic acid, benzoic acid, acetic acid, ascorbic acid, succinic acid, nicotinic acid, formic acid, maleic acid, and so forth. Since tetracycline is amphoteric, salts of various metallic elements with the antibiotic may be prepared, e.g. sodium, potassium, calcium, magnesium. The alkali metal salts of tetracycline are formed by treating an aqueous suspension of the antibiotic with two equivalents of an alkaline hydroxide. The solid metal salts of tetracycline are obtained by the evaporation under vacuum of an aqueous solution of the antibiotic at the proper pH. Preparation of tetracycline from neutral solution, as by lyophilization, produces the free base, generally as a trihydrate if water is present and heat is not applied. Tetracycline trihydrate is also prepared by dissolving the base in a Water-soluble organic solvent, e.g. methanol, ethanol, and pouring the resulting solution into water. Solid calcium tetracycline is converted to tetracycline trihydrate by solution 'in aqueous acid, e.g. to pH 3 or less using sulfuric acid or hydrochloric acid, followed by the addition of an agent to tie up the calcium, e.g., sodium Versenate (ethylenediamine-tetraacetic acid), and finally by addition of an alkali to raise the pH to at least 6 but not substantially above pH 8 whereupon tetracycline trihydrate precipitates. Tetracycline trihydrate is recrystallized from aqueous alcohols, e.g., methanol, and is converted to the anhydrous form by the application of moderate heat in vacuo over a drying agent.

The acid addition salts of tetracycline are prepared in various ways. For example, stoichiometric amounts of acid are added to a solution of tetracycline base in water or an organic solvent, e.g., butanol, acetone, methanol, to form the acid addition salt; when desired the solid salt is then isolated by lyophilizing the resulting mixture (as in water), or by collecting the precipitated salt by filtration (as from ethyl acetate), or by precipitating the salt by the addition of another organic solvent (as in the addition of a hydrocarbon, e.g., Skellysolve, to butanol or cyclohexane to ethyl acetate), or by reducing the volume of solvent by distillation in vacuo until the salt precipitates upon cooling (e.g., from butanol). Many other methods are used to prepare these salts, e.g., tetracycline is converted to tetracycline hydrochloride by adsorption of tetracycline on a chromatographic adsorbent (e.g., a silica such as Florisil) followed by elution of tetracycline with acid, e.g., aqueous or methanolic or butanolic hydrochloric acid. In another method tetracycline base in water having pH about 8-9 is extracted into butanol; the butanol is separated and the tetracycline base is converted to tetracycline hydrochloride and trans- 'ferred to an aqueous phase by extraction with aqueous hydrochloric acid; the tetracycline hydrochloride so prepared may be isolated if desired, e.g., by lyophilization. In one preferred method, tetracycline base is converted to tetracycline salts by dissolving the base in dry acetone or n-propanol and adding the anhydrous acid, i.e., hydrogen chloride gas, sulfuric acid, sulfuric acid in dry acetone, phosphoric acid, tartaric acid, citric acid, nitric acid in dry methyl isobutyl ketone, and collecting by filtration the salt which precipitates, e.g., tetracycline hydrochloride or the sulfate, phosphate, tartrate, citrate, etc.

EXAMPLE 10 Tetracycline is readily extracted from alkaline fermentation liquors by non-polar organic solvents. The following procedure has been used.

One liter of Streptomyces viridifaciens fermentation broth at pH 8.5 is extracted with 0.5 liter methyl isobutyl ketone. The organic solvent is separated, washed with water, reduced by azeotropic distillation to a volume of about 20 mls. and added to 250 mls. Skellysolve C. Twenty mgm. of tetracycline (lot 26) precipitate and are collected by filtration and found to have an activity at one mgm./ml. diluted 1:64 of 19.5 mm. (zone diameter 12 by B. subtilis plate assay on pH 6.2 agar); 24.2 mm. at 1:16 and 27.7 mm. at 1:4. The original broth assayed at 27.3 mm. at 1:4 dilution.

EXAMPLE 11 In another procedure, 4.5 liters of Streptomyces viria'ifaciens fermentation broth at pH 6.7 are adjusted to pH 5.5 and extracted with 3 liters butanol. The butanol extract is separated, washed with water, reduced in volume to about 35 mls. by azeotropic distillation and added to 350 mls. Skellysolve C (technical heptanes). Tetracycline (lot 27) precipitates as a solid and is collected by filtration (1.2 grams, having an activity at one mgm./ml. diluted 1:16 of 23.2 mm.; zone diameter by B. subtilis plate assay agar at pH 6.2).

The residual broth at pH 5.5 after extraction with butanol is adjusted to pH 8.5 and extracted with 3 liters butanol. The butanol is separated, washed with water, reduced in volume to about 30 mls. and added to 350 ml. Skellysolve C. Crude tetracycline (lot 28) precipitates as an orange-yellow solid having an activity at one mgm./ml. diluted 1:16 of 25.2 mm. (zone diameter of B. subtilis plate assay on pH 6.2 agar). The original broth assayed 27.3 mm. at 1:4 dilution.

EXAMPLE 12 In a third procedure 2.5 liters of Streptomyces viridifaciens fermentation broth assaying 25.7 mm. at 1:4 dilution is adjusted to pH 6.4 and extracted with 2 liters butanol. The butanol is separated, washed with water, reduced in volume to about 50 ml. by azeotropic distilla tion and added to one liter Skellysolve C. Dark yellow, solid tetracycline (500 mgm.) (lot 24) precipitates and is collected by filtration and found to have an activity at 1 mg./ml. diluted 1:16 of 18 mm. (zone diameter by B. subtilis plate assay on agar at pH 6.2) and 23 mm. at 1:4 dilution.

The broth at pH 6.4 after butanol extraction is kept in the cold room for three days, and then adjusted to pH 8.5 with sodium hydroxide and extracted with 2 liters butanol. The butanol is separated, washed with water, reduced in volume to about 30 ml. by azeotropic distillation and added to 600 ml. Skellysolve C. Dark brown, solid tetracycline mg.) (lot 25) precipitates and is collected by filtration and found to have an activity at one mg./ml. diluted 1:16 of 28 mm. (zone diameter by B. subtilis plate assay on pH 6.2 agar).

EXAMPLE 13 In another procedure 760 m1. of Streptomyces viridifaciens fermentation broth adjusted to pH 9.4 with sodium hydroxide is extracted with 380 ml. n-butanol. The nbutanol is separated, added to aqueous hydrochloric acid to give pH 7.35, and the butanol is removed by vacuum distillation; the residual aqueous concentrate is made up to about 38 cc. with water, and lyophilized to yield 339 mgm. solid tetracycline, which is found to have an activity at one mgjml. buffer of 25 mm. (zone diameter by B. subtilis plate assay on pH 6.2 agar) and of 18.7 mm. at 1:3 dilution. The original broth assayed 26.3 mm. (and 20.0 mm. at 1:3 dilution) and the spent broth assayed 19.0 mm. (and 12.7 mm. at 1:3 dilution).

EXAMPLE 14 In another procedure 5.5 liters of Streptomyces viridifaciens fermentation broth adjusted to pH 5 .5 is extracted with 3 liters n-butanol. The solvent is separated, washed with water, reduced in volume to about 35 rnls. by azeotropic distillation and added to 350 mls. Skellysolve C. Golden yellow, solid tetracycline (1.2 g.) precipitates and is collected by filtration as solid A and found to have an activity at l mgm./ml. diluted 1:16 of 23.2 mm. (zone diameter by B. subtilis plate assay on agar at pH 6.2) and 26.9 mm. at 1:4 dilution.

The residual broth, after butanol extraction at pH 5.5

is adjusted to pH 8.5 and extracted with 3 liters of butanol'. The butanol is separated, washed with water, reduced in volume to about 30 mls. by azeotropic distillation and added to 350 mls. Skellysolve C (technical heptanes).

14 having R values of 1 to inclusive. A small spot in the exact center would thus have a value of R 6 while a larger spot would extend into the adjacent zones and have R 5, 6, 7 as the entire zone is counted.

Additional orange-yellow, solid tetracycline (0.5 g.) (lot 5 Using this technique, the R spectra of tetracycline 30) precipitates and is collected by filtration as solid B (lot 30) is found to be as follows, using 5 microliters of and found to have an activity at l mgm./ml. diluted 1:16 a 1 mgm./ml. solution of the antibiotic as a sample for of 25.2v mrn. (zone diameter by B. subtilis assay on agar each strip and assaying with B. subtilis in pH 6.2 agar, at pH 6.2) and 28.5 mm. at 1:4 dilution. for the strips developed in the twelve solvent systems:

Solvent System A B O D E F G H I .T K L Rrvalue 6,7 5,6 4,5 4,5,6 1 8,9,10 6,7 8 5 9,10 6,7,8 3,4,5

Tetracycline is conclusively characterized, even when The composition of these solvent systems is as follows: contaminated by similar organic chemicals by its spec- AWater trum of activity (i.e., degree of migration or R values) B10% aqueous sodium citrate using a series of solvents in the procedure known as C-40% aqueous sodium citrate paper strip chromatography. This technique isarelatively D-Butanol saturated with water new but already Wellestablished procedure for the identi- E-Dry methyl isobutyl ketone fication of organic compounds; as with infra-red maxima FA mixture of 100 parts of 80% methanol and 10.5 the R values in a series of solvent systems are a unique parts piperidine adjusted to pH 9.5 with acetic acid. and reproducible characteristic of a given chemical and GA mixture by volume of 80 parts methanol, 5 parts serve as a fingerprint. glacial acetic acid and 15 parts Water.

The procedure used is as follows. Strips of ash-free, H-Butanol saturated with water and containing 2% dense, highly retentive filter paper (e.g., 589 Blue Ribp-toluenesulfonic acid. bon Special from Carl Schleicher and Schuell Co., Keene, IA mixture of 100 mls. of butanol saturated with N.H.), half inch wide and 58 cm. long, are suspended at Water and 5 ml. glacial acetic acid. constant room temperature in a protected area (i.e., in a IButanol saturated with water and containing 2% large jar) from the edge of a dish. The top of each strip p-toluenesulfonic acid and 2% piperidine. is in contract with a supply of particular solvent system Kl00 parts of butanol saturated with water and 2 (also called developing phase) in the dish; each of the grams p-toluenesulfonic acid plus 2 mls. piperidine plus strips is suspended from a different dish containing a dif- 2 grams lauric acid. ferent one of the plurality of solvent systems used in the LA mixture of two parts isoamyl alcohol and one test; the bottom of each strip hangs free and does not reach part chloroform, saturated with 10% aqueous sodium the supply of its solvent system (or volatile components citrate. In this case, before applying the sample, the thereof) placed below the hanging strip to. facilitate satu- Strips are saturated with 10% aqueous sodium citrate ration of the air with the solvent in use. adjusted to pH 5.7 with citric acid and dried.

The product to be examined (i.e., in a fermentation broth or as an isolated solid) is placed on a marked spot EXAMPLE 15 at the top of that portion of the strip which hangs free Solid A from EXBIIIIP1e 14 is extractfid With 50 m1. of in the air. In the case of a solid, it is dissolved in any aqueous ammonium hydroxide of P leaving behind useful solvent. The amounts used are those giving acon- 220 mgmsof brown soiids- Solid B from venient zone size on final assay as determined by simple Example 14 is extracted With 30 of aqueous trial. For example, a useful amount is 5 microliters moiiium hydroxide of P 64,1eflvihg behind 120 mgms- (0.005 ml.) of a solution containing 1 mgm./ml. of tetraof Solids- The basic, aqueous Solutions of tetracycline cycline to solvent for subsequent assay vs. B. subtilis. are combined and Passed through a Chromatographic The strip is dried and then placed With its upper end in umii by containing Fiorisil (Chromatoposition in the dish, Which contains the selected solvent graphic From tho brown ffl nt there is resystem, Th solvent i ll d to migrate downward, covered about 600 mgm. of brown solids of low activity. that is, to develop the strip, until the solvent front reaches The column iS then eluted With 25 mls. acetone and the bottom of the strip. This requires about fifteen hours; then 125 ethahoi and the yellow ehlatos are the surrounding atmosphere is maintained at a constant carded- The column is then eluted With 1% hydrochloric temperature free from drafts and saturated with solvent acid The acid ethanol l a is flashed into vapor f a 13001 below water (i.e., distilled in vacuo with the addition of water Each Strip is h removed, ailudried and placed on a until all the organic solvent has been removed) and the tray of agar of controlled pH (here 6.2) inoculated with a Water is neutralized With amhlollillm Y id and then test organism, in this case B. subiilz's. After standing in iyophiiized to Yield Solid tetracycline (l t 32) Weighing the refrigerator overnight, the strips developed by the difgram and having an acitiviiy at 1 g d l t d ferent solvent systems are removed, and the trays are 1316 of 24 (Zone diameter by ubtili-S' plate assay marked for identification and location of the strips, inon agar at P cubated overnight and either read directly or photo- 65 EXAMPLE 6 graphed to 5 a Permanent record- In the manner. of Example 15 above Florisil-adsorbed g j g g g g gg fi p iy v on broth is extracted with pH 8.5 butanol and the eluate tion if each antibiotic a t e tlil o gi o o dried by azeotropic distillatlon. On pouring into the g h 6 Strip i marked y Skellysolve C,, there is precipitated and recovered solid a clear area, WhlCh contrasts W1th the turbid area where tetrac 11 500 1 the test organism has rown The stri a h ye e 0t 36) having an actmty at the photograph is maried into s i zl g g al'ngnh/litall. gilutgcll lzlf of greater than 30 mm. (zone 10$ 5 0, 10 and 15 percent of the me er y "ispate assay on agar at P distance from the point of sample application to the EXAMPLE 17 bottom, respectively, and these zones are described as Tetracycline of any purity is converted to the sodium salt by treating the material in water with sodium hydroxide until the pH is over 9.5. The solution is then frozen and dried under vacuum to yield the dry sodium salt in the form of a stable, water-soluble powder.

EXAMPLE l8 Streptomyces BL 456667A fermentation broth [500 Solvent System Sample 6667-1-11; value 6667-2-Rg value ml. assaying 16.9 mm. versus S. Aureus undiluted (X) and 13.8 mm. diluted three-fold (3X)] prepared according to the above methods was adjusted with sodium hydroxide to pH 9.08 and extracted with 250 m1. n-butanol. The butanol was separated and adjusted to pH 6.90 by adding water and hydrochloric acid. The butanol was removed from this mixture by distillation in vacuo, leaving an aqueous concentrate of tetracycline, which was made up to 25 ml. by the addition of Water, assaying 19.5 mm. versus S. aureus diluted three-fold, and then lyophilized to yield 115 mgm. solid tetracycline (lot 6667A- 3) assaying 19.4 mm. versus S. aureus at 1 mgm./ml.

An additional 500 ml. of this broth was adjusted to pH 2.37 with phosphoric acid and extracted with 250 EXAMPLE 20 The distribution coeflicient of tetracycline between butanol and water (concentration in butanol divided by concentration in water) is found to be less than one at any pH but to be altered markedly by the addition of calcium ion. Thus, the distribution coeflicient of tetracycline between butanol and 2.0% aqueous calcium chloride is less than 2 (and often less than 1) from pH 1 to about 6.6 but rises sharply with increasing pH and is greater than 5 from about pH 7.4 to 11, reaching a maximum of 9-10 between pH 9 and 10. A very similar pattern of tetracycline distribution coeflicient versus pH is obtained using butanol and 0.5% aqueous calcium chloride, with a maximum of about 20 at ml. n-butanol. The butanol was separated, adjusted to about PH 10. NO distribution coefiicient for tetra pH 6.40 wit]:1 gvater taid sodlum laydrdoxlgie. T e butanol cycline greater than 1.5 is obtained between butanol i fi mm s mature f 1st ii g i ii and aqueous solutions containing five or ten percent eavma an aqueous concentrate f? tetracyc He w sodium sulfate or two, five, or ten percent sodium chlo- 1 3 1 3 21 1 162238 a l-1: xiggl gggtg i h r e rf l c l fi ride from pH 4 to 9. Distribution coefiicients between r an 2 and 4 can be obtained with 210% NaCl only at the then lyophlllzed E Yleld 356 solld tetracycllfle very acidic pH 2, which renders commercial operation 6667A-4) assaying 15.1 mm. versus S. aureus at 1 d fli lt although not impossible, mgIIL/ Extraction of an aqueous solution (broth) containing d The spectra f these p f 0f tetracyflllle Was 40 at least 250 meg/ml. tetracyclme with one-quarter etermlned as described above, using S. aureus, to be as volu e of butanol at pH 8.5 m the presence of 0.5% follows: calcium chloride yielded a high recovery (40-50%) of Solvent System A B C D E F 7 8 G Sample 6667A3Rr value 4-7 5,6 1-5 3,4,5 1 8,9,10 6,6 8,9,10 7,8,9 6667A-4-R;value--- 5,6 5,6,7 3,4,5 4, 5,6 1 8, 9, 1o 9,10 6-9 Solvent system 7 was 80% methanol containing 2% 5O tetracycline as solids of good potency after separation piperidine, 2% p-toluenesulfonic acid and 2% lauric acid. and concentration of the butanol, back-extraction into Solvent system 8 was pyridine containing 1% water, 1% aqueous acid, such as hydrochloric, and isolation therep-toluenesulfonic acid and 1% lauric acid. from. Tetracycline is then extracted as described herein.

19 mass? at t ar -r ises;

s 1 e r0 e.g., 1 e

streptomyfies BL 456567 farmefltatlon' broth 410 liquor) as well as of added calcium ion. The calcium assaylng g-" dwells undlluted a ion may be added as any salt, e.g., calcium chloride, muted flhfiefold (3 X) PTePPTed aFCOI'dmg calcium carbonate, and preferably use is made of a total to the above methods was ad usted with sodium hyconcentration of calcium ion in the broth in the range g fi i f extract? 1 of 0.01 to 2.0 percent. Addition of about 0.1 percent g g gb gg g g i fi gg i fg e calcium chloride is a valuable procedure; the amount butanol was removed from this mixture by distillation 2 f be i neiceslsary ig m in vacuo, leaving an aqueous concentrate of tetracycline e rot t e stole lomefnc equlva em 0 e tetra' which was made up to 21 ml. by the addition of water, cyclme P as determined 9 assay of i broth for assaying 18.0 mm. diluted three-fold and then lyophilized tetracyflme and calclum Excesslve f t to yield 93 mgrn. solid tetracycline (lot 6667-4) assayof calcium 1on at times are not desirable as they precipiing 193 versus 5 am-eus at 1 L tate calc1um phosphates which tend to absorb tetra- An additional 410 m1. of this broth was adjusted to cycllne; this can be recovered but only at the expense pH 2.59 with phosphoric acid and extracted with 205 of additional labor.

ml. n-butanol. The butanol was separated and adjusted Additional information on the increased efliciency of extraction of tetracycline into butanol from fermentation broth containing calcium ion is furnished by the observation that the addition of a sequestering agent for calcium ion to a broth reduced the extraction of Tetracycline present in meg/ml.

Portion Percent In Buta- In Spent n01 Broth EXAMPLE 21 To a fermentation broth containing at least 100 meg/ml. of tetracycline there is added about 0.1% CaCl and the broth is adjusted to about pH 8.5-9.0 with caustic or ammonia and then extracted with about onefifth volume of n-butanol by multiple stage extraction. The mixture is filtered and the tetracycline-containing butanol is concentrated by distillation in vacuo with the optional addition of sufficient water at the end to permit the formation of hydrates. Tetracycline solids (at least 600 mcg./mgm.) precipitate as base or calcium salt and are collected by filtration and dissolved in minimal amounts of n-propanol. Gaseous hydrogen chloride is then passed into the n-propanol to precipitate crystalline tetracycline hydrochloride of approximately theoretical potency.

EXAMPLE 22 To a tetracycline-containing fermentation broth there is added 0.1% CaCl and the broth is adjusted to about pH 8.5-9.0 with caustic, e.g. soda or potash, or ammonia and then extracted with n-butanol, using /2 volume for batchwise extraction or 4- volume for multiple stage extraction. The mixture is filtered and the butanol phase is separated if batchwise procedure is used. The tetracycline containing butanol is concentrated to of the original broth volume by distillation in vacuo. The butanol concentrate and any insoluble therein are extracted with three successive equal volumes of water adjusted to pH-2.5 with hydrochloric acid. These aqueous extracts are combined, filtered and concentrated by distillation in vacuo to broth volume. To this aqueous concentrate is added 1% calcium chloride (w/v) and the pH is adjusted to about 7.8-8.5 by the addition of ammonium hydroxide. The resulting precipitate of solid calcium tetracycline is collected by filtration and dried with an acetone wash and air or vacuum drying.

EXAMPLE 23 Tetracycline free base (one gram) is dissolved in a minimum volume of anhydrous acetone and the stoichi'- ometric amount of hydrogen chlo-ride required to form the salt is passed in as a gas. Solid, crystalline yellow tetracycline hydrochloride precipitates and is collected by filtration.

EXAMPLE 24 Tetracycline is purified of contaminating amounts of chlortetracycline or oxytetracycline, when such are present, by countercurrent distribution, as in the Craig apparatus, using a system of n-butanol versus 2.5% acetic acid, as described, for example, in Weisberger, Technique of Organic Chemistry, vol. III, Interscience Publ. Co., N.Y., 1950, pages 171-311, and Anal. Chem., vol. 24, pages 66-70 (1952), and vol. 23, pages 41-44 (1951).

18 EXAMPLE 25 Cationic, anionic and nonionic wetting agents are added to tetracycline fermentation broths to increase the efficiency of extraction of tetracycline from the broths into butanol or amyl alcohol or methyl isobutyl ketone. The pH is adjusted carefully in each case to obtain the maximum distribution into the organic solvent as determined by simple test. Examples of useful agents and concentrations are Aerosol OT (1.0%), Tween 21 (0.5%), and Tergitol (0.1%). Concentration used may beas high as 5%. Other useful agents include anionic wetting agents such as lauric acid, stearic acid, esters of sulfuric acid, naphthalene sulfonic acids, higher alkyl aromatic sulfonic acids; cationic wetting agents such as quaternary ammonium salts (e.g.,

where OCR represents mixed fatty acids of coconut oil); and nonionic wetting agents such as alkyl phenol polyglycol ethers.

In one simple procedure by this method, a carrier is used to transfer the tetracycline from fermentation broth, containing at least meg/ml. of tetracycline, to methyl isobutyl ketone. This solvent is separated; the addition of gaseous hydrogen chloride precipitates solid crystalline tetracycline hydrochloride.

In another procedure by this method, tetracycline is carried from broth into methyl isobutyl ketone by the addition to the broth of pH 2-4 of about 0.2% of an anionic wetting agent such as Tergitol 4 (sodium tetradecyl sulfate), Tergitol 7 (sodium heptadecylsulfate) or Aerosol OT (dioctyl sodium sulfosuccinate).

EXAMPLE 26 A very simple, inexpensive and efficient method of isolating crude solid tetracycline from large volumes of fermentation broth is furnished by the following procedure. A fermentation broth containing at least 250 meg/ml. tetracycline, and preferably 1000 meg/ml. tetracycline, or more, is adjusted to an acid pH and the mycelium is removed by filtration. The filtered broth is then adjusted to pH 8 or higher by the addition of caustic, e.g. soda, or ammonia. Crude, active, solid tetracycline precipitates as the free base or calcium salt, contaminated with calcium phosphates and other impurities, and is collected by filtration. This crude product is further purified, if desired, by the use of procedures described above. For example, this product is then dissolved in hydrochloric acid of pH 2 containing 10% sodium chloride, extracted into butanol and the butanol is separated and concentrated by distillation in vacuo to precipitate tetracycline hydrochloride.

EXAMPLE 27 Several batches of tetracycline were prepared from Streptomwces viridifaciens fermentation broths in the following manner: To whole broth (-285 gallons) there was added 285-532 g. calcium chloride and 50-120 gallons wet n-butanol. The pH of the broth, which was 4.9-5.5, was adjusted to 835-88 with 50% sodium hydroxide (4-l0.5 liters). After stirring, which gave an emulsion separated by filtration using Dicalite (diatomaceous earth), the butanol phase containing tetracycline was separated (100-122 gallons) and concentrated to a volume of about 2.5-7.0 liters by distillation in vacuo at liquid temperature of less than 100 F. and under less 19 then adjusted to about.6.0 .with ammonium hydroxide, precipitating tetracyclinewhich was collected-by filtration. A second crop of tetracycline was frequently obtained by concentrating the filtrate again to about onefifth volume.

The various batches of tetracycline obtained above (63 g.) were combined and dissolved in 630 ml. water adjusted to pH 1.5 with hydrochloric acid. To this solution after filtration there was added 63 g. Versene Acid (ethylenediaminetetraacetic acid). The pH was then adjusted to 6.0 with ammonium hydroxide and the tetracycline which precipitated (30 g.) was collected by filtration, dried in .vacuo over P and slurried twice with 120 ml. methanol, discarding 14.5 g. insoluble material. The methanol solutions were combined and flashed into 400 ml. water. Tetracycline precipitated from this aqueous solution upon standing overnight indthe cold room and was collected by filtration (8.9g. in twocrops) and air-dried. v I The tetracycline was treated with .carbon while in solution in water at pH 1.5; the solution was filtered and thetetracycline precipitated by raising the-pH to 3.5 with ammonium hydroxide. The tetracycline (6.9 g.) was dissolved in 34.5 ml.; water adjusted to pH 1.5 with by- .drochloric acid; the solution-was adjusted to pH 3.5 with ammonium hydroxide and seeded; crystalline hydrated tetracycline base slowly precipitated and was collected by filtration (6.45 g.), air-dried and found to melt at about 171.5173.5 C., to assay 890 mcg./mgm. by ultraviolet analysis (based on 1000 mcg./mgm. tetracycline hydrochloride or 964 mcg./mgm. tetracycline trihydrate) and 892 meg/mgm. by bio-assay versus B. subtilz's on pH 8.0 plates and to be free of chlortetracycline and oxytetracycline or other impurities as shown by paper strip chromatography. This crystalline hydrated tetracycline base contained 19.2% moisture and was analyzed after drying. I

Analysis.-Calculatcd for C H O N Calculated Found 0 59.45 59.6 H 5. 44 5.45 N e 6. 6.29

This crystalline hydrated tetracycline base (2 grams) i was dissolved in 20 ml. n-propanol containing about 0.5 ml. concentrated hydrochloric acid. After standing thirty minutes at room temperature and ninety minutes in the cold, crystalline tetracycline hydrochloride pre cipitated and was collected by filtration (1.5' g.) and found to melt at 217-219 C. with decomposition.

Analysis.-Calculated for C H O N HCl:

I Calculated Found 54. 9 55. 0 a a 5. 99 Q1 7. 37 7. o

A suspension obtained by mulling this crystalline tetracycline hydrochloride in mineral oil (Nujol) shows many characteristic absorption bands in the infrared. Among these are the following frequencies (in reciprocal centimeters): 3340, 1678, 1623, 1597, 1315, 1248, 1229, 1175,

743, 719, 692, and 667. The infrared absorption spectrum of this mineral oil mull within the charactersitic region of wave numbers between 1350 and 650 cm." is shown in Fig. 2 of the accompanying drawings.

Tetracycline has [a] 245 at a concentration of 1% in methanol and shows ultraviolet absorption peaks at 267 mu (5, 17,400) and 355 my. (6, 13,500) in 0.1 N hydrochloric acid. Tetracycline hydrochloride has [a] "253 at a concentration of 0.5% in 0.1 N hydrochloric acid.

EXAMPLE 2:;

Tetracycline is isolated from fermentation broths by acidifying the broth to about pH 2.0-2.5, e.g. with sulfuric acid, and filtering. The clear filtrate containing the tetracycline is then adjusted to about pH 9-10 with alkali, e.g. sodium hydroxide or ammonia. Some sodium carbonate may be added near the end to react with calcium ion present, thus precipitating calcium carbonate which adsorbs tetracycline. The tetracycline which precipitates at .pH 910 has a potency of at least 200 mcg./mgm. and is collectedby filtration and purified according to the methods described above.

As an example of such purification, this product is dissolved in aqueous nitric acid at pH 2, sodium nitrate is added and the tetracycline is extracted with n-butanol. The butanol is separated and neutralized, precipitating tetracycline base.

In another example, this product is slurried directly with n-butanol, from which pure tetracycline is recovered, e.g. as hydrochloride, upon acidification and concentration.

In a third method, this product is slurried in dilute sulfuric acid, filtered and the filtrate adjusted to about pH 6 by adding alkali, thus precipitating solid, purified tetracycline.

Either the filtered, acid broth or the alkaline filtrate from the precipitation of tetracycline from broth are suitable for use as collected or after drying as an animal feed or additive therefor. These filtrates contain large amounts of valuable food factors (e.g. proteins, carbohydrates, minerals, vitamins, and especially vitamin B and congeners, unidentified stimulants for animal growth, often called animal protein factors) and may be added to animal feeds, particularly those of vegetable origin, either before or after concentration or drying. The value of such animal feeds and additives from these filtrates is increased by the addition to the medium before fermentation of'a soluble cobalt salt, e.g. about 0.1 to 20 parts per million of the nutrient medium of cobalt nitrate, and by the addition to the medium before fermentation of a substance (such as an alkali metal cyanide or an alkali metal ferrocyanide or ferricyanide) furnishing a source of cyanide ion in an amount within the range of about 0.1 to parts per million.

EXAMPLE 29 An eifective method of obtaining crystalline tetracycline from fermentation broths is furnished by the following procedure. Broth is acidified to about pH 2.0-2.5, e.g. with sulfuric acid, filtered and the solids are discarded. The filtrate is adjusted to an alkaline pH, e.g. about 9.0- 9.5, and extracted with one-third volume of n-butanol. The butanol is separated, discarding the aqueous phase (spent broth), acidified with sulfuric acid, e.g. to pH 5-6, and concentrated by distillation in vacuo to about one-fiftieth of the original broth volume. The tetracycline which precipitates is collected by filtration and found to have a potency of at least 200 mcg./mgm. The butanol is recycled. The crude tetracycline (100 g.) is dissolved in one liter of water acidified to about pH 1.5 with sulfuric acid; insoluble matter is removed by filtration and discarded. The aqueous solution is treated with activated carbon (10-50 g.) and filtered.- At this point it is optional to add about 1 0 g. Sequestrene (ethylenediamine-tetraacetic acid) or another agent to sequester cal-' ciurn. The pH of this aqueous solution is then adjusted to about 3.5, e.g. with ammonia, and on standing pure tetracycline base crystallizes out and is collected by filtration.

Additional less pure tetracycline suitable for re-cycling is precipitated from the final filtrate by raising the pH to about 6.

Sequestering agents useful for removing calcium and thus facilitating precipitation of pure tetracycline base include citric acid, tartaric acid, sodium phytate, gluconic acid, phosphates such as sodium metaphosphate and in general the members of this class known to the art, e.g. see J. Chem. Educ. 25, 482-488 (1948) and references therein. Many of these agents are weakly ionized, polybasic acid compounds or hydroxylated, organic carboxylic acids. I

EXAMPLE 30 An effective and simple method of isolating tetracycline from fermentation broths without the use of large volumes of solvents for extraction is furnished by the following procedure. The broth is adjusted to pH 9-10 by the addition of alkali and filtered. Less than 100 meg/ml. tetracycline remains dissolved in the filtrate, which is discarded. This mixture with mat (mycelium) of adsorbed and precipitated tetracycline and other broth constituents is suitable for use as collected or after drying as an animal feed or additive therefor. The filtrate, which contains little tetracycline but large amounts of valuable food factors (e.g. proteins, carbohydrates, minerals, vitamins and especially vitamin B and congeners, unidentitied stimulants for animal growth, often called animal protein factors) may also be added to animal feeds, particularly those of vegetable origin, either before or after concentration or drying. The value of such animal feeds and additives from both precipitate and filtrate is increased by the addition to the medium before fermentation of a soluble cobalt salt, e.g. about 0.1 to 20 parts per million of the nutrient medium of cobalt nitrate, and of a substance (such as an alkali metal cyanide or an alkali metal ferrocyanide or ferricyanide) furnishing a source of cyanide ion in an amount Within the range of about 0.1 to 100 parts per million.

The solid mat and adsorbed tetracycline is extracted at low pH (e.g. 1.5) with small volumes of aqueous acid, e.g. sulfuric acid, oxalic acid, to give a solution containing at least 3000 meg/ml. tetracycline.

In one method the tetracycline is isolated by neutralizing this acid solution to pH 4-10, and preferably at least pH 6, thus precipitating solid tetracycline, in base or calcium form, of a potency of at least 100 mcg./mgm.

In another method, the tetracycline is isolated by neutralizing this acid solution to pH 9-10 and extracting the tetracycline with about one-third volume of n-butanol. The butanol is separated and concentrated to about onetwentieth volume, precipitating tetracycline, in base or calcium form, of a potency of at least 200 mcg./mgm.

EXAMPLE 31 Crude damp tetracycline base (e.g. 1 g., 20% water) is conveniently converted to crystalline hydrochloride by dissolving the base in a minimum amount of dry n-butanol (e. g. ml.) by the addition of concentrated hydrochloric acid (e.g. 0.3 ml), filtering and decolorizing with carbon if desired, and then adding an additional amount of dry butanol (e. g. 5 ml) Pure tetracycline hydrochloride crystallizes out on standing and is collected by filtration. The first filtration may be omitted; in that case all the butanol is added at once and the tetracycline dissolves and then crystallizes out spontaneously as tetracycline hydrochloride.

EXAMPLE 32 Tetracycline is purified of contaminating amounts of chlortetracycline, when present, by slurrying the free base 22' in methanol. The chlortetracycline base does not dissolve; the dissolved tetracycline base is recovered in purified form as above, e.g. by evaporation of the solvent or by pouring the methanol into water.

EXAMPLE 33 Tetracycline is efficiently purified of contaminating amounts of chlortetracycline, when such are present, by slurrying in aqueous acid of substantially pH 2.5, which dissolves only the chlortetracycline. The preferred acid is hydrochloric acid; other acids, e.g. sulfuric acid, phosphoric acid, may be used, however, provided the requisite pH is obtained. Tetracycline base or salt is slurried in hydrochloric acid sufficient to provide a pH of 2.5 or, as is preferred, impure tetracycline base or salt, e.g. hydrochloride, is dissolved in aqueous acid at pH 1.5 or less and sufiicient base, e.g. ammonium hydroxide, sodium hydroxide, is added to provide a pH of 2.5. The amount of tetracycline used is not a limiting factor; in general it is convenient to use 50 to 100 mgm. tetracycline base or salt per milliliter of water acidified to pH 2.5 with hydrochloric acid. The pH is a critical factor; substantially pH 2.5 is preferred although any pH in the range 2.0-3.5 is useful. The amount of chlortetracycline present as an impurity in tetracycline to be purified by this procedure, should not exceed 9 1ngm./ml. of acidified water and preferably shouldbe less than 5 mgm./ml.; this limit is easily observed by increasing when necessary the amount of acid water used for slurrying. Such solids, e.g. tetracycline hydrochloride and chlortetracycline hydrochloride, in this purification are thus mixed, i.e., slurried, in water acidified with hydrochloric acid to substantially pH 2.5 for a considerable period of time, e.g. sixteen hours, sufficient to permit the chlortetracycline to dissolve. This is determined by simple test. The purified, solid tetracycline base which remains undissolved is then collected by filtration.

Thus, tetracycline hydrochloride (10 g.) assaying 830 incg/mgm. tetracycline and 171 n1cg./mgm. chlortetracycline (83% tetracycline) by the differential ultraviolet absorption method and containing about 10% chlortetracycline as determined by paper-strip chromatography, was slurried for sixteen hours in 200 ml. water adjusted to about pH 2.5 with hydrochloric acid. The solid, purified tetracycline base was then collected by filtration, dried, found to weigh 8.0 grams, to assay 901 1ncg./mgm. tetracycline and 39 mcg./mgm. chlortetracycline (96% tetracycline) by the differential ultraviolet absorption method and to contain -97% tetracycline as determined by paper-strip chromatography. The recovery of tetracycline was 87%.

A similar experiment conducted under nitrogen gave a 92% recovery of tetracycline base containing from zero to three percent chlortetracycline, potency 929 mcg./mgm.

EXAMPLE 34 Tetracycline is efficiently purified of contaminating amounts of chlortetracycline, when such are present, by slurrying in aqueous alkali of about pH 7.5-9.0, which dissolves only the chlortetracycline. In another embodiment, the base or acid addition salt is dissolved at pH 9.5 and precipitated as purified tetracycline base by adjusting the pH to 7.59.0, preferably about 8.5, by the addition of acid, e.g. hydrochloric acid. The preferred alkali is ammonia but other water soluble alkalies, such as sodium hydroxide or triethylamine, are useful provided that they furnish the requisite pH. Tetracycline base or acid addition salt, e.g. hydrochloride, is slurried in water adjusted to about pH 8.5-9.0 by the addition of alkali. The amount of tetracycline is not a limiting factor; in general it is convenient to use 50 to mgm. per ml. water. The pH is a critical factor and must be sub stantially pH 7.59.0. The amount of chlortetracycline, present as an impurity in the tetracycline to be purified by this procedure, should not exceed 12 mgm./m1. of alkaline water and preferably should be less than 2 mgm./ml.; this limit is easily observed by increasing when necessary the amount of alkaline water used for slurrying. Such solids, e.g. tetracycline and chlortetracycline, in this purification are thus mixed, i.e. slurried, in water made alkaline with ammonium hydroxide to substantially pH 8.5-9.0 for a considerable period of time, e.g.- sixteen hours, sufiicient to permit the chlortetracycline to dissolve. This is determined by simple test. The purified, solid tetracycline base which remains undissolved is then collected by filtration. The entire operation is preferably conducted in an oxygen-free atmosphere, e.g. under nitrogen, to minimize or eliminate decomposition.

Thus, tetracycline hydrochloride (10- g.) assaying 945 mcg./mgm. tetracycline and 171 mcg./mgm. chlortetracycline (85% tetracycline) by the differential ultraviolet absorption method and containing about 10% chlortetracycline as determined bypaper-strip chromatography, was slurried under nitrogen for sixteen hours in 200 ml. water adjusted to pH 9.0 with ammonium hydroxide. The solid, purified tetracycline base was then collected by filtration, dried, found to weigh 8.5 g., to 'assay 986 mcg./mgm. tetracycline and 20 mcg./mgm. chlortetracycline (98.1% tetracycline) by the difierential ultraviolet absorption method and to contain 9798% tetracycline as determined by paper-strip chromatography. The recovery of tetracycline was 89%.

All assays refer to equivalent micrograms of tetracycline hydrochloride. Thus, the theoretical maximum potencies in mcg./mgm. for anhydrous solids are tetracycline hydrochloride 1000 and tetracycline base 1080.

EXAMPLE 35 Calcium tetracycline is purified by extracting with methanol in which it is insoluble, and then dissolving in methanol containing calcium chloride. Undissolved impurL ties are removed by filtration and water and ammonia to pH Sare added to the solution to precipitate purified calcium tetracycline.

EXAMPLE 36 Tetracycline of any purity may be converted to the hydrochloride by treating the material in water with hydrochloric acid until a clear solution is obtained and the pH is below 3-4. The solution is frozen and dried under vacuum to give a readily soluble powder.

EXAMPLE 37 Calculated Found H20, none lost at 60? O. in vacuo.

The Omegamycin of the present invention has been found to be the same substance which is now commonly designated tetracycline, whose properties are described in the Journal of the American Chemical Society, volume 75, pages 4621-23, 1953. 1

Thus, a sample of tetracycline free base was prepared. according to the literature from chlortetracycline and found to melt at .l70175"- C. andto contain 0.71 percent chlorine indicatingacontamination with about percent unreacted chlortetracycline. :This sampleand another, chlorine-free sample of pure tetracycline .(of M.P. 169-17l 'C. with decomposition) were shown to be the same as a sample of Omegamycin (lot 30; prepared according to Example 14 above) and all impurities were distinguished, 'by examination of these samples and samples of chlortetracycline and oxytetracycline, both alone and as mixtures, by paper strip chromatography (particularly using solvent systems D and L).

That Omegamycin (lot 30) is tetracycline was also shown by a finding of identical rate of loss of activity in pH 8.0 buffer at 37 C. at about 0.7 mgm./ml. (28% loss in 48 hours); chlortetracycline lost about 50% of its activity in 14 hours and oxytetracycline lost about 50% of its activity in 26 hours under the same conditions.

In addition, Omegamycin is clearly. differentiated from chlortetracycline and oxytetracycline by assay on pH 8.0 agar, on which chlortetracycline and oxytetracycline appear much less active than Omegomycin.

Assays of solids were performed on B. subtilis plates with agar pH 6.2 and pH 8.0 and using buffers described above, as diluent, corresponding to the agar pH. The results of typical runs follow:

Tetracycline may thus be purified of contaminating amounts of chlortetracycline or oxytetracycline by maintaining an aqueous solution at about pH 8.0 until all the chlortetracycline or oxytetracycline is decomposed and then isolating purified tetracycline, as above.

Thus, solid tetracycline base containing about 26% chlortetracycline was dissolved in aqueous sodium hydroxide and additional sodium hydroxide was added to raise the concentration to 0.17-1.17 Normal. After standing at room temperature or 40 C. for 6, 20, or 45 minutes, the solution was acidified to pH 1.5-2.0, filtered and the tetracycline precipitated and recovered by rais ing the pH of the filtrate to 3.5 or 6.0. The recovered tetracycline was of good potency and contained less than two percent chlortetracycline.

In an additional case about 45,000 meg/ml. of crude tetracycline was freed of contaminating chlortetracycline by solution for 24 hours at pH 8.5 at room temperature and subsequent recovery.

The tetracycline (Omegamycin) of the present invention is useful as the free base, including its anhydrous and hydrated forms and particularly the trihydrate, in combating many diseases caused by bacterial infection in man and animals. For this use, tetracycline is associated with a significant amount of a pharmaceutically acceptable carrier which may be either a solid material or a liquid. The compositions may take the form of tablets, efiervescent tablets, powder, granules, capsules (both hard and soft shell capsules), or suspensions in edible oils, or other dosage forms which are particularly useful for oral ingestion. Liquid diluents are employed in sterile conditions for parenteral use, that is, by injection. Such a medium may be a sterile solvent or suspending agent such as Water or an injectable oil. The compositions may take the form of active material, namely tetracycline (which is used in this discussion to refer to the free base or hydrates thereof), admixed-With solid diluents and/or tabletting adjuvants such as cornstarch, lactose, talc, stearic acid, magnesium stearate, gums or the like. Any of the encapsulating or tabletting materials used in pharmaceutical practice may be employed Where there is no incompatibility with the tetracycline. The materials may be tabletted with or without adjuvants. Alternatively, the tetracycline may be placed in the usual capsule or resorbable material such as the usual gelatin capsule and administered in that form. In yet another embodiment, tetracycline may be put up in powder packets and so employed. Tetracycline may be prepared in the form of a palatable suspension in which the tetracycline is not soluble, e.g., coconut oil. In this case, use may be made of coconut oil modified to have a setting point below 60 F. and/or gelled with an aluminum stearate. Such a suspension may be given orally as made or may be encapsulated. Tetracycline ointments and lotions are useful topically; use for topical therapy is made of nose drops, troches, and suppositories. The tetracycline of the present invention is particularly useful when given by the oral or intramuscular routes; a useful dosage range in man is about 10-1000 mgms. per dose. Dosages are given about one to six times per day, depending on the patient, the infection, the route of administration and the like.

The percentage of active ingredients in these compositions may be varied. It is necessary that the active ingredients constitute a proportion such that a suitable dosage will be obtained. Obviously, several unit dosage forms may be administered at about the same time. Although it is found, particularly on intravenous injection; that a percentage of less than 0.10 percent of tetracycline is effective, it is preferable to use not less than 0.10 percent of tetracycline. Activity increases with the concentration of tetracycline. The percentage of active agent may be 10 percent, or 25 percent, or even a higher proportion of the substance administered. For example, tablets may be prepared with a minor proportion of diluent and a major proportion of active material. Tablets containing from about 10 to 1000 mgms. of tetracycline are particularly useful. The solid pharmaceutical carrier used may be an envelope enclosing pure tetracycline, e.g. pure tetracycline trihydrate in a gelatin capsule.

The solubility of tetracycline in water between pH and 7 is very low (approximately 0.4 mgm./ml.). The addition of calcium chloride, e.g., 1% w./v., enables much more concentrated solutions to be obtained having a solubility greater than 5 mgm./ml., e.g. at pH 6 or in the range of pH 5-7. These solutions are highly viscous, although the viscosity may be altered by pH adjustment within the range of high solubility. Stable solutions or suspensions with higher tetracycline content are thus readily obtained which are of value in pharmaceutical formulations.

Highly concentrated aqueous solutions of tetracycline in excess of 20 mgm./ml. at pH 3.5-9 suitable for oral and parenteral use are obtained by mixing tetracycline, a nontoxic heavy metal salt, e.g. aluminum sulfate, and tartaric acid or similar sequestering agents such as citric acid, ethylenediarnine-tetra-acetic acid, pluconic acid and phytic acid. By heavy metal is meant any metal other than the alkali metals; thus alkaline earth metals such as 26 calcium are included within the present definition of heavy metals. A similar composition of like utility and solubility is prepared by mixing a sequestering agent and a heavy metal salt of tetracycline.

While various embodiments of the invention have been described in some detail, it will be understood that various modifications may be made in the procedures described and the products disclosed without departing from the scope of the invention. Certain agents, compounds, or mixtures (e.g. acids, media, solvents, and the like) and other details described in relation to one example or description of procedure may be employed in connection with other examples or procedures.

We claim:

1. A process for recovering tetracycline from a fermentation broth comprising the consecutive steps of adjusting the pH of an :sidic fermentation broth to a pH in the range of 8.5 to 9.4 inclusive, extracting the tetracycline with n-butanol, separating the n-butanol and recovering the tetracycline therefrom.

2. A process for recovering tetracycline from a fermentation broth comprising the consecutive steps of adjusting the pH of an acidic fermentation broth to a pH in the range of about 8.5 to 9.0, inclusive, extracting the tetracycline with n-butanol, separating the n-butanol and recovering the tetracycline therefrom, said broth at the time of n-butanol extraction containing about 0.1 percent of calcium chloride added after the end of fermentation.

References Cited in the file of this patent UNITED STATES PATENTS 2,482,055 Duggar Sept. 13, 1949 2,516,080 Sobin July 18, 1950 2,586,766 Pidacks Feb. 19, 1952 2,609,329 Niedercorn Sept. 2, 1952 2,640,842 Weidenheimer June 2, 1953 2,655,535 Pidacks Oct. 13, 1953 2,656,347 Goett Oct. 20, 1953 2,658,078 Blase Nov. 3, 1953 2,699,054 Conover Jan. 11, 1955 2,734,018 Minieri Feb. 7, 1956 2,739,924 Lein et al Mar. 27, 1956 2,804,476 Bogert Aug. 27, 1957 FOREIGN PATENTS 506,950 Belgium Nov. 30, 1951 497,345 Canada Nov. 3, 1953 OTHER REFERENCES Weidenheimer: Abstract of Serial No. 83,780, 650 0.6. 895, Sept. 18, 1951.

Van Dyck et al.: Antibiotics and Chemotherapy, vol. 2, April 1952, pp. 184-198.

Antibiotics and Chemotherapy, vol. 4, pp. 375-379; 750-752.

Martin: Antibiotics Annual, 1954-1955 (Abstract 156), pp. 1020-1024.

Ann., N.U. Acad. Sci., 60, Art. 1, pp. 86-95.

Baldacci. Archiv. fur Mikrobiologie, Classification of the Actinomyces Species, vol. 20, No. 4 (1954), pp. 347-357. 

1. A PROCESS FOR RECOVERING TETRACYCLINE FROM A FERMENTATION BROTH COMPRISING THE CONSECUTIVE STEPS OF ADJUSTING THE PH OF AN ACIDIC FERMENTATION BROTH TO A PH IN THE RANGE OF 8.5 TO 9.4 INCLUSIVE, EXTRACTING THE TETRACYCLINE WITH N-BUTANOL, SEPARATING THE N-BUTANOL AND RECOVERING THE TETRACYCLINE THEREFROM. 